PROJECT SUMMARY Doxorubicin is a well-established and highly effective chemotherapy drug commonly used to treat multiple cancer types, but its use is limited by cardiotoxicity. Cardiotoxicity can range from asymptomatic reduction in left ventricular ejection fraction to highly symptomatic heart failure (Class III to IV). Acute doxorubicin-induced cardiotoxicity (DIC) occurs in ~11% of patients and long-term cardiotoxic side effects are observed in up to 36% of patients. However, the underlying mechanisms of DIC remain largely unknown, hampering the development of effective therapeutics for DIC. To that end, in this proposal we aim to use state-of-the-art approaches in genomics and epigenetics to identify the genetic and molecular mechanisms of DIC. In Aim 1, we will generate iPSC lines from 100 cancer patients treated with doxorubicin, 50 of whom experienced cardiotoxicity and 50 did not. From these lines, we will perform RNA-seq and eQTL mapping to discover novel single nucleotide polymorphisms (SNPs) responsible for DIC, and the relevant SNPs will be introduced to or deleted from non- DIC or DIC patient iPSC lines, respectively, with the CRISPR gene editing technique. We will investigate the functional and transcriptional changes and determine whether the identified SNP is responsible for the disease phenotype. In Aim 2, we will perform a combination of epigenetic techniques in ATAC-seq, ChIP-seq, and IP- mass spectrometry to identify genes regulated by topoisomerase II-beta (TOP2B), a transcriptional regulator known to be inhibited by doxorubicin. We hypothesize that TOP2B controls genes critical for cardiomyocyte contraction, metabolism, and homeostasis, the expression of which is disrupted upon TOP2B inhibition by doxorubicin. Results from these experiments will reveal the specific genes regulated by TOP2B, which can be used as potential therapeutic targets of DIC. In Aim 3, we will identify genes suitable as drug targets that are related to DIC using the CRISPR genome screening approach. This novel technique offers a unique and cost- efficient opportunity to systemically screen for drug target genes dysregulated by doxorubicin treatment in iPSC- CMs. Using patient-specific iPSC-CMs, the proposed aims will allow us to elucidate for the first time the genetic and molecular basis for DIC.